319
Clinica Chimica Acta, @ Elsevier/North-Holland
87 (1978)
319-326
Biomedical
Press
CGA 9347
GLUCOSES-PHOSPHATASE ACTIVITY IN LIVER AND BLOOD PLATELETS OF TWO PATIENTS WITH GLYCOGEN STORAGE DISEASE TYPE I
YOGO OKA a**, TAKASHI MITSUYAMA a, BUNSAKU NAGAI ARASHIMA a, IWAO OHKUBO a and ICHIRO MATSUDA b
a, SHINICHIRO
a Department of Paediatrics, Hokkaido University School of Medicine, Sapporo (Japan) and b Department of Paediatrics, Kumamoto University Medical School, Kumamoto (Japan) (Received
March
22nd,
1978)
Summary Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The KM value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.
Introduction Glycogen storage disease type I (von Gierke’s disease) is one of the most common types of glycogenosis and is known to be due to glucose-6-phosphatase (EC 3.1.3.9) deficiency [l]. Although clinical symptoms and several biochemical tests are useful for a conjecture of the disease, only enzyme analysis can confirm the diagnosis. Absence of G-6-Pase activity in liver, kidney and intestinal mucosa is evidence for the diagnosis of GSD type I [l-3]. Low activity of G-6-Pase in liver was found in some cases of GSD type I [1,4,5]. Kinetic enzyme studies on the low G-6-Pase activity of such cases have not been
* Address: Y. Oka. Department of Paediatrics, Hokkaido University School of Medicine, North 14 West 5, SaPPOrO. Japan. Present address: Y. Oka, Department of Paediatrics. Laboratory of Developmental Biochemistry, University of Groningen. Bloemsingel 10. Groningen. The Netherlands.
320
reported. It is not confirmed whether this low activity represents nonspecific phosphatase activity or incomplete defects. Recently, blood platelets (material easily available as opposed to liver) have been used for confirmation of the diagnosis [6-lo]. But it seemed to be difficult to detect platelet G-6-Pase because of its low activity as compared to the activity in the liver and because of the presence of nonspecific phosphatase. We had an opportunity to study two patients suspected of GSD. A low activity of G-6-Pase in liver of both patients was found. The Michaelis constant (K,) for the G-6-P of the residual activity of liver in two patients was similar to that found in human control liver. But we could not demonstrate any difference of platelet G-6-Pase activity between our patients and controls. These findings conflict with the previous reports, in which a reduced activity of platelet G-6Pase in patients with GSD type I is demonstrated [6-lo]. Case reports Case 1, U.K. was a 5$-year-old Japanese girl who was admitted to our University Hospital because of abdominal protuberance noted soon after birth by her parents. Nasal bleeding had occurred frequently since 2 years of age. She was short in stature, but appeared well-nourished. She had doll-like facies. Physical examination revealed a large smooth, firm liver (about 10 cm below costal margin). One episode of hypoglycemia (blood glucose 40 mg/dl) with ketonuria was found after overnight fasting. Laboratory studies were not remarkable except for the findings summarized in Table I. The results of tolerance tests are shown in Table II. At open biopsy, her liver showed a yellow, fatty appearance. Microscopically, the hepatic cells were large with increased amounts of carbohydrate granules by PAS staining. There were many clear areas, reflecting loss of fat, within the hepatic cells. Case 2, J.M. was a 3&-year-old Japanese girl who has transferred to our University Hospital from others. She had a large liver and was suspected of GSD. Histologically, her liver demonstrated properties similar to those of case 1. Also she had clinical and physiological abnormalities similar to those found in case 1; hepatomegaly, short stature, doll-like facies, nasal bleeding, hypoglycemia with ketonuria, hyperlactic acidemia, hyperlipemia, hyperuricemia, slight glucose response to glucagon and some abnormalities of platelet functions (Tables I and II). Materials and methods Open liver biopsy was performed on the two patients on the same day during admission. A control liver was obtained from a female adult suffering from echinococcosis by laparotomy at the same time as the patient’s open biopsy. The other two control liver samples (a male of 6 months of age with congenital heart disease, a lo-days-old premature female infant with respiratory distress syndrome) were taken within 120 min after death. These liver samples were immediately frozen and stored at -80°C until enzyme assay. The liver homogenate was prepared in ice-cold 0.25 M sucrose solution. G-6-Pase activity in liver homogenate was determined by the method of Baginski et al. [ 111, except
321
for the measurement of the liberation of inorganic phosphate. This was carried out by the Fiske-Subbarow method [ 121. The residual homogenates were centrifuged for 30 min at 11000 X g in a refrigerated centrifuge and the supernatant was centrifuged for 60 min at 105 000 X g. The precipitate (microsome) was suspended in ice-cold 0.25 M sucrose, 1 mM EDTA solution, pH 7.0. G-6Pase activity at various substrate concentrations was determined by the method of Baginski et al. [ 111. 25 mM /3-glycerophosphate (P-GLP) was used as a substrate to determine the nonspecific phosphatase. Glycogen content of liver was determined by the use of anthrone reagent [ 131. For collecting platelets, 10 ml of blood was taken from two patients and 10 or 200 ml from control adults by venipuncture, mixed with 0.5 ml of 0.06 M EDTA in 0.8% NaCl solution per 10 ml of blood and centrifuged at 350 X g at 4°C for 20 min. The supernatant was recentrifuged at 350 X g for 10 min, preventing contamination of leukocytes and red blood cells. The supernatant was again centrifuged at 900 X g at 4°C for 20 min. The precipitated platelets were washed twice with 10 ml of 0.03 M EDTA in 0.9% NaCl solution, and finally were suspended in 0.5 ml 0.03 M EDTA, 0.9% NaCl solution. The platelets were disrupted by ultrasound and were used for the enzyme assay. Platelet G-6-Pase activity was measured by the method of Schrijver et al. [lo], in which 25 mM G-6-P with or without added fl-GLP was used as a substrate. The protein concentrations of the platelet used for the enzyme assay ranged from 1.0 to 6.4 mg/ml assay medium. Liberated glucose was measured by the glucose oxidase method [14]. Leukocytes were collected by the method described earlier [ 151. Amylo-1,6-glucosidase activity in leukocytes was assayed by the method of Williams [ 161. Phosphorylase activity in the liver tissue and leukocytes was determined by the method of Williams and Field [ 171. Protein determinations were done by the method of Lowry et al. [18]. Results The laboratory findings obtained during admission are shown in Table I. Both patients demonstrated hypoglycemia after overnight fasting, hyperlactic acidemia, hyperuricemia, increasing concentrations of FFA and &lipoprotein and slight elevation of SGOT and SGPT. Further, the patients exhibited abnormalities of platelet functions, including prolongation of bleeding time, decrease of platelet adhesiveness and abnormal aggregation reactions. Table II shows the glucose response to glucagon, alanine and galactose tolerance tests. In both patients only a slight elevation of blood glucose levels occurred after glucagon administration and the hyperglycemic response to alanine and galactose could not be elicited. True G-6-Pase activity was obtained by subtracting the nonspecific phosphatase activity from the total phosphatase activity. Table III shows that the two patients had reduced activities of true G-6-Pase in liver homogenate, 17% and 6% of control liver in case 1 and case 2, respectively. Both patients showed the increase of glycogen content in liver and had normal activities of phosphorylase and amylo-1,6-glucosidase in liver or leukocytes. Fig. 1 and 2 show Lineweaver-Burk plots with G-6-P as a substrate. This assay was carried out with the liver microsomes of the two patients and the controls. An apparent
322
TABLE
I
LABORATORY
FINDINGS Case
Fasting Blood
blood
glucose
lactate
Case
1 (U.K.)
2 (J.M.)
NOrmal
4049
38-54
60-100
23.5-35.0
23.144.9
9-16
mg/dl mg/dl
Serum
uric acid
9.0-l
7.4-10.2
2.4-5.4
Serum
cholesterol
222-318
162-231
150-250
Serum
triglyceride
390434
590-940
65-170
2.46
1.42
0.5
>2.9
>4.2
1.2-2.2
68-264
5-40
79-145
3-35
Plasma Serum
FFA P-lipoprotein
2.4
SGOT
97-l
SGPT
132-184
Bleeding
time
10
Platelet
adhesiveness
Platelet
aggregation
ADP
(2
X 10-e
Epinephrine
(5
M) X 10-h
M)
Collagen
TABLE
RESPONSE
Glucagon
(30
galactose
TO
j.tg/kg
(2 g/kg,
body
1
2
TABLE
mequiv./l mm
5
l-3
14.3
17.2
60-80%
29.0
43.0
58.3
f 16.1%
68.0
40.0
82.6
r
5.3%
30.0
65.0
82.6
r
5.3%
orally)
GLUCAGON, weight,
were
ALANINE
AND
intramuscularlu).
administered Glucose
Case
mg/dl mg/dl
min
(Duke)
II
GLUCOSE
Case
70
mg/dl
after
10% overnight
concentration
GALACTOSE alanine
TOLERANCE
solution
(0.5
g/kg,
TESTS intravenously)
fasting.
(mg/dl)
0
30
60
Glucagon
69
66
90
Alanine
57
58
57
Galactose
76
70
70
90
120
150
180
95
76
79
50
66
64
56
57
74
73
72
79
Glucagon
68
80
90
81
61
57
57
Alanlne
55
51
55
56
59
61
51
Galactose
41
49
39
42
39
42
39
min
III
GLYCOGEN
CONTENT
AND
ENZYME
ACTIVITIES
Glycogen
G-6-Pase
content
(nmol
(g/loo wet
g
Pi/mg
proteinlmin)
in liver homogenate
Phosphorylase
Amylo-1,6-
(pm01
glucosidase
Pi/
g proteinlmin)
weight)
in liver
(cpmlglmin) in leukocyte
Total
Nonspecific
True
Liver
Leukocyte
Case
1 (U.K.)
7.1
4.57
0.86
3.71
216
50.5
3248
Case
2 (J.M.)
7.9
3.72
2.37
1.35
160
43.9
3940
22.50
1.00
21.0-31.5
2132-9687
Control
(adult)
Control
(N = 10)
21.50
120
and
323
Fig. 1. Lineweaver-Burk case 2.
plots of G+Pase
in liver microsomes
of two patients.
l-
0, case 1; o------O,
Fig. 2. Lineweaver-Burk plots of G-6-Pase in liver microsomes of human control livers. Control 10 days (autopsy); control 2. female adult (biopsy); control 3, male 6 months (autopsy).
TABLE
IV
GLUCOSE-6-PHOSPHATASE The values of the patients
ACTIVITY represent
IN BLOOD
the mean of three determinations.
G-6-Pase activity
Case 1 Case 2 Control (N = 10) Mean ? S.E. Range
PLATELETS
(nmol
glucoselmg
G-6-P
G-6-P (25 mM), fl-GLP (50 mM)
24.1 38.3
18.1 20.6
50.7 ? 5.9 20.1-70.4
25.7 ? 2.4 19.9-39.9
protein/h)
1. female
324
K, for G-6-P of approximately 7.7 mM can be calculated both for the two patients and the controls. The maximum velocities (V,) were 12.5 and 5.3 nmol per mg protein per min for case 1 and case 2, respectively. The control livers yielded values of 91, 133 and 222 nmol per mg protein per min. Nonspecific phosphatase in liver microsomes was not detectable. As shown in Table IV, no significant difference in platelet G-6-Pase activity was found between our patients and. controls when assayed at 25 mM G-6-P with or without 50 mM /3-GLP, though the activities of the patients were close to the low levels of the controls. Discussion Our two patients had clinical and physiological abnormalities similar to those of GSD type I or type III. Usually low fasting blood glucose and mild elevation of blood lactate and cholesterol have been noted in GSD type III. But marked hyperlipemia and nasal bleeding accompanying the abnormalities of platelet function as found in our patients are characteristic manifestations of GSD type I. Also the results of tolerance tests suggest a G-6-Pase deficiency. Reduced G-6-Pase activity, normal activities of phosphorylase and amylo1,6-glucosidase, and elevated glycogen content indicate that both patients are suffering from GSD type I. The slight response to glucagon seems to correlate with low activity of G-6-Pase in liver of both patients [2]. The low activity of G-6-Pase prompted a further characterization of this activity. An apparent K, for G-6-P of 7.7 mM for both of the patients and for controls was found (Fig. 1 and 2) This is of same order as found for human or other mammalian G-6Pase [ 19-201. We could not demonstrate other characteristics of the residual activity because of limiting amounts of material. Further investigations are necessary to show whether this low activity of G-6-Pase represents an incomplete defect or a mutant enzyme. We found a high nonspecific phosphatase activity in the liver of case 2 as found in some cases of GSD type I. The patients with GSD type I reported by Field had high activities of nonspecific phosphatase with @-GLP as a substrate in both liver and intestinal mucosa. These activities were comparable to those as found with G-6-P as a substrate [31-
TABLE
V
PLATELET
GLUCOSE-6.PHOSPHATASE
References
ACTIVITY
G-B-Paw
activity
(nmol
REPORTED gIucose/mg
Controls Linneweh Negishi Schrijver
* The
et al. [91
Substrate
? 432
*
0
-210
*
G-6-P
k 192
*
47
-112
*
G-6-P
0.27
i -i
245
0.05
0.03-
40*
16
20
study
25.1 are calculated
Patients
156
et al. [lo]
values
protein/h)
403
et al. [81
Stormont
Present
et al. [61 et al. [71
Soyama
IN LITERATURE
as 0.6
X 108
?
2.4 platelets
[U-‘4ClG-6-P
*
G-6-P,
0.9.1.7
G-6-P
+ P_GLP
G-6-P
+ ~3-GLP
18.1, are equal
0.07
20.6
to 1 mg of protein.
G-l-P
32.5
Several investigators have reported that G-6-Pase is present in human platelet and that the determination of platelet G-6-Pase activity can be used for the diagnosis of GSD type I [6-lo]. The carrier detection of the disease using platelets was also shown by Soyama et al. [ 71, Negishi et al. [8] and Stormont et al. [ 91. Previous reports are summarized on Table V. The values reported for the control range from 0.27 to 756 nmol per mg protein per hour. Our values for the control are consistent with those of Schrijver et al. [lo], though its activity is much lower than found by Linneweh et al. [ 61, Soyama et al. [7] and Stormont et al. [9]. Their values for the control (245--756 nmol per mg protein per hour) correspond to more than A of G-6-Pase activity (53 nmol Pi liberated per mg protein per min) in normal liver [21]. It seems unlikely that human platelet contained such high G-6-Pase activity, because gluconeogenesis was not present in the way established for liver and kidney. Schrijver et al. demonstrated very low or absent activities of FDPase, PEPCK and PC in human platelets [lo]. In addition the enzyme assay in the cases of Linneweh et al. [6], Soyama et al. [ 71 and Negishi et al. [8] was performed without correction for nonspecific phosphatase, using only G-6-P as a substrate. Stormont et al. [ 91 were using G-1-P for measuring nonspecific phosphatase, and reported this activity was insignificant in more than half of the normal controls while some controls and his patient showed definite levels of nonspecific phosphatase. But Schrijver et al. suggested that the use of G-1-P was not correct because of the presence of glucose phosphomutase in platelet [lo]. Schrijver et al. reported that platelet G-6-Pase activities in his two patients were of the same order of magnitude as those of normal controls when assayed with only G-6-P as a substrate, whereas the enzyme activities assayed in the substrate mixture of G-6-P and fl-GLP were extremely low in the two patients, and concluded that the glucose released from G-6-P in the presence of P-GLP at high concentration could be considered as G-6-Pase activity [lo]. We could not demonstrate any significant difference of platelet G-6-Pase between our patients and control, though the addition of /3-GLP took care of nonspecific phosphatase. In our patients 50 mM fl-GLP might block some of the activity of nonspecific phosphatase but some of this enzyme might remain active upon G-6-P. In view of this, we added increasing concentrations of /I-GLP
TABLE THE
VI EFFECTS
BLOOD The
OF
P-GLP
ON
GLUCOSE-6-PHOSPHATASE
AND
NONSPECIFIC
values
in parentheses
represent
liberated
phosphate
with
only
fl-GLP
as a substrate
tein/h). Substrate G-6-P
25 mM 25 mM
PHOSPHATASE
IN
PLATELETS
Control
Case
1
case
+ @-GLP
+ 12.5
mM
46.7 41.4
21.8 (53.5)
36.4 -
25mM+25mM
41.4
(89.3)
-
-
25mM+50mM
23.8
(143.0)
13.1
17.5
25mM+125mM
21.4
(154.5)
10.9
5.8
25mM+250mM
0.0
(345.0)
0.0
0.0
2
(-01
Pi/mg
pro-
326
to the assay medium. As shown in Table VI, the glucose released from 25 mM G-6-P was decreased by raising the concentration of P-GLP both in two patients and control, and showed about half of that assayed without fl-GLP, when enzyme assay was performed in the substrate mixture of 25 mM G-6-Z’ with 50 mM P-GLP. The liberated phosphate increased by raising concentration of fl-GLP when assayed in only P-GLP as a substrate in control, suggesting the presence of definite levels of nonspecific phosphatase activity. However no glucose production was obtained at a concentration of 250 mM P-GLP. This indicated that the inhibition by high P-GLP of G-6-Pase might occur. Further, we could not deny the possibility that the glucose production (P-GLP 50 mM and 125 mM) indicated the hydrolysis of G-6-P by nonspecific phosphatase, not by G-6-Pase, because it has not yet been confirmed that G-6-Pase is present in human platelet. Our observations indicate that this assay method is not suitable for the proof of diagnosis of GSD type I. We recommend to determine the platelet G-6-Pase activity of many other patients with GSD type I for reaching a definite conclusion. Acknowledgements The authors are grateful to Dr. F.A. Hommes and Dr. J. Schrijver for many discussions and help in the preparation of the manuscript. This investigation was supported by a Research Grant aided by the Ministry of Health and Welfare of Japan for the Research on Handicapped Children, 1977. References 1 Cori, G.T. and Cori, C.F. (1952) J. Biol. Chem. 199.661-667 2 Cornblath, M. and Schwartz, R. (1976) in Disorders of Carbohydrate Metabolism in Infancy (Schaffer. A.J. and Markowitz, M., eds.). pp. 237-260, W.B. Saunders Company, Philadelphia 3 Field, J.B.. Epstein, S. and Egan. T. (1965) J. Clin. Invest. 44.1240-1247 4 SokaI. J.E.. Lowe, C.U., Sarcione, E.J., Mosovich, L.L. and Doray, B.H. (1961) J. CIin. Invest. 40, 364-374 5 Stamm, W.E. and Webb, D.I. (1975) Arch. Intern. Med. 135.1107-1109 6 Linneweh. F., L&r, G.W., Wailer, H.D. and Gross, R. (1962/63) Enzymol. Biol. CIin. 2.188-195 7 Soyama. K.. Schimada, N., Kusunoki. T. and Nakamura, T. (1973) CIin. Chim. Acta 44,327-331 8 Negishi, H.. Morishita, Y.. Kodama, S. and Matsuo. T. (1974) CIin. Chim. Acta 53, 175-181 9 Stonnont. D., Davies, C. and Emmerson. B.T. (1976) Clin. Chim. Acta 71, 303-308 10 Schrijver. J.. Koster. J.F. and Hiilsmann. W.C. (1975) Eur. J. Clin. Invest. 5, 7-14 11 Baginski, E.S.. Foa, P.P. and Zak, B. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U.. ed.), pp. 876-880, Academic Press, New York 12 Fiske, C.H. and Subbarow, Y. (1925) J. Biol. Chem. 66.375-400 13 Hassid. W.Z. and Abraham, S. (1957) in Methods in Enzymology, Vol. III, PP. 34-50. Academic Press, New York 14 Glucomesser direct kit was purchased from Tokyo Zoki Chemical Companv 15 Oka. Y., Matsuda. I., Arashima, S.. Anakura. M.. Mitsuyama. T. and Nambu. H. (1975) NeuropPdiatrie 6.202-209 16 WiBiams, H.E., Kendig, E.M. and Field, J.B. (1963) J. CIin. Invest. 42, 666-660 17 WiIhams. H.E. and Field J.B. (1961) J. CIin. Invest. 40.1841-1845 18 Lowry. O.H., Rosebrough. N.J., Far+. A.L. and RandalI, R.J. (1951) J. Biol. Chem. 193.265-275 19 Nordlie, R.C. (1976) in Gluconeogenesis. its Regulation in Mammahan Species (Hanson, R.W. and Mehhnan. M.A.. eds.), pp. 93-152, Wiley-Interscience. New York 20 Rezani. I. and Duffy, J.L. (1971) Biochem. Med. 5. 237-244 21 Hefferan, P.M. and HowelI. R.R. (1977) Biochim. Biophys. Acta 496. 431-435
Clinica Chimica Acta, @ Elsevier/North-Holland
87 (1978)
319-326
Biomedical
Press
CGA 9347
GLUCOSES-PHOSPHATASE ACTIVITY IN LIVER AND BLOOD PLATELETS OF TWO PATIENTS WITH GLYCOGEN STORAGE DISEASE TYPE I
YOGO OKA a**, TAKASHI MITSUYAMA a, BUNSAKU NAGAI ARASHIMA a, IWAO OHKUBO a and ICHIRO MATSUDA b
a, SHINICHIRO
a Department of Paediatrics, Hokkaido University School of Medicine, Sapporo (Japan) and b Department of Paediatrics, Kumamoto University Medical School, Kumamoto (Japan) (Received
March
22nd,
1978)
Summary Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The KM value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.
Introduction Glycogen storage disease type I (von Gierke’s disease) is one of the most common types of glycogenosis and is known to be due to glucose-6-phosphatase (EC 3.1.3.9) deficiency [l]. Although clinical symptoms and several biochemical tests are useful for a conjecture of the disease, only enzyme analysis can confirm the diagnosis. Absence of G-6-Pase activity in liver, kidney and intestinal mucosa is evidence for the diagnosis of GSD type I [l-3]. Low activity of G-6-Pase in liver was found in some cases of GSD type I [1,4,5]. Kinetic enzyme studies on the low G-6-Pase activity of such cases have not been
* Address: Y. Oka. Department of Paediatrics, Hokkaido University School of Medicine, North 14 West 5, SaPPOrO. Japan. Present address: Y. Oka, Department of Paediatrics. Laboratory of Developmental Biochemistry, University of Groningen. Bloemsingel 10. Groningen. The Netherlands.
320
reported. It is not confirmed whether this low activity represents nonspecific phosphatase activity or incomplete defects. Recently, blood platelets (material easily available as opposed to liver) have been used for confirmation of the diagnosis [6-lo]. But it seemed to be difficult to detect platelet G-6-Pase because of its low activity as compared to the activity in the liver and because of the presence of nonspecific phosphatase. We had an opportunity to study two patients suspected of GSD. A low activity of G-6-Pase in liver of both patients was found. The Michaelis constant (K,) for the G-6-P of the residual activity of liver in two patients was similar to that found in human control liver. But we could not demonstrate any difference of platelet G-6-Pase activity between our patients and controls. These findings conflict with the previous reports, in which a reduced activity of platelet G-6Pase in patients with GSD type I is demonstrated [6-lo]. Case reports Case 1, U.K. was a 5$-year-old Japanese girl who was admitted to our University Hospital because of abdominal protuberance noted soon after birth by her parents. Nasal bleeding had occurred frequently since 2 years of age. She was short in stature, but appeared well-nourished. She had doll-like facies. Physical examination revealed a large smooth, firm liver (about 10 cm below costal margin). One episode of hypoglycemia (blood glucose 40 mg/dl) with ketonuria was found after overnight fasting. Laboratory studies were not remarkable except for the findings summarized in Table I. The results of tolerance tests are shown in Table II. At open biopsy, her liver showed a yellow, fatty appearance. Microscopically, the hepatic cells were large with increased amounts of carbohydrate granules by PAS staining. There were many clear areas, reflecting loss of fat, within the hepatic cells. Case 2, J.M. was a 3&-year-old Japanese girl who has transferred to our University Hospital from others. She had a large liver and was suspected of GSD. Histologically, her liver demonstrated properties similar to those of case 1. Also she had clinical and physiological abnormalities similar to those found in case 1; hepatomegaly, short stature, doll-like facies, nasal bleeding, hypoglycemia with ketonuria, hyperlactic acidemia, hyperlipemia, hyperuricemia, slight glucose response to glucagon and some abnormalities of platelet functions (Tables I and II). Materials and methods Open liver biopsy was performed on the two patients on the same day during admission. A control liver was obtained from a female adult suffering from echinococcosis by laparotomy at the same time as the patient’s open biopsy. The other two control liver samples (a male of 6 months of age with congenital heart disease, a lo-days-old premature female infant with respiratory distress syndrome) were taken within 120 min after death. These liver samples were immediately frozen and stored at -80°C until enzyme assay. The liver homogenate was prepared in ice-cold 0.25 M sucrose solution. G-6-Pase activity in liver homogenate was determined by the method of Baginski et al. [ 111, except
321
for the measurement of the liberation of inorganic phosphate. This was carried out by the Fiske-Subbarow method [ 121. The residual homogenates were centrifuged for 30 min at 11000 X g in a refrigerated centrifuge and the supernatant was centrifuged for 60 min at 105 000 X g. The precipitate (microsome) was suspended in ice-cold 0.25 M sucrose, 1 mM EDTA solution, pH 7.0. G-6Pase activity at various substrate concentrations was determined by the method of Baginski et al. [ 111. 25 mM /3-glycerophosphate (P-GLP) was used as a substrate to determine the nonspecific phosphatase. Glycogen content of liver was determined by the use of anthrone reagent [ 131. For collecting platelets, 10 ml of blood was taken from two patients and 10 or 200 ml from control adults by venipuncture, mixed with 0.5 ml of 0.06 M EDTA in 0.8% NaCl solution per 10 ml of blood and centrifuged at 350 X g at 4°C for 20 min. The supernatant was recentrifuged at 350 X g for 10 min, preventing contamination of leukocytes and red blood cells. The supernatant was again centrifuged at 900 X g at 4°C for 20 min. The precipitated platelets were washed twice with 10 ml of 0.03 M EDTA in 0.9% NaCl solution, and finally were suspended in 0.5 ml 0.03 M EDTA, 0.9% NaCl solution. The platelets were disrupted by ultrasound and were used for the enzyme assay. Platelet G-6-Pase activity was measured by the method of Schrijver et al. [lo], in which 25 mM G-6-P with or without added fl-GLP was used as a substrate. The protein concentrations of the platelet used for the enzyme assay ranged from 1.0 to 6.4 mg/ml assay medium. Liberated glucose was measured by the glucose oxidase method [14]. Leukocytes were collected by the method described earlier [ 151. Amylo-1,6-glucosidase activity in leukocytes was assayed by the method of Williams [ 161. Phosphorylase activity in the liver tissue and leukocytes was determined by the method of Williams and Field [ 171. Protein determinations were done by the method of Lowry et al. [18]. Results The laboratory findings obtained during admission are shown in Table I. Both patients demonstrated hypoglycemia after overnight fasting, hyperlactic acidemia, hyperuricemia, increasing concentrations of FFA and &lipoprotein and slight elevation of SGOT and SGPT. Further, the patients exhibited abnormalities of platelet functions, including prolongation of bleeding time, decrease of platelet adhesiveness and abnormal aggregation reactions. Table II shows the glucose response to glucagon, alanine and galactose tolerance tests. In both patients only a slight elevation of blood glucose levels occurred after glucagon administration and the hyperglycemic response to alanine and galactose could not be elicited. True G-6-Pase activity was obtained by subtracting the nonspecific phosphatase activity from the total phosphatase activity. Table III shows that the two patients had reduced activities of true G-6-Pase in liver homogenate, 17% and 6% of control liver in case 1 and case 2, respectively. Both patients showed the increase of glycogen content in liver and had normal activities of phosphorylase and amylo-1,6-glucosidase in liver or leukocytes. Fig. 1 and 2 show Lineweaver-Burk plots with G-6-P as a substrate. This assay was carried out with the liver microsomes of the two patients and the controls. An apparent
322
TABLE
I
LABORATORY
FINDINGS Case
Fasting Blood
blood
glucose
lactate
Case
1 (U.K.)
2 (J.M.)
NOrmal
4049
38-54
60-100
23.5-35.0
23.144.9
9-16
mg/dl mg/dl
Serum
uric acid
9.0-l
7.4-10.2
2.4-5.4
Serum
cholesterol
222-318
162-231
150-250
Serum
triglyceride
390434
590-940
65-170
2.46
1.42
0.5
>2.9
>4.2
1.2-2.2
68-264
5-40
79-145
3-35
Plasma Serum
FFA P-lipoprotein
2.4
SGOT
97-l
SGPT
132-184
Bleeding
time
10
Platelet
adhesiveness
Platelet
aggregation
ADP
(2
X 10-e
Epinephrine
(5
M) X 10-h
M)
Collagen
TABLE
RESPONSE
Glucagon
(30
galactose
TO
j.tg/kg
(2 g/kg,
body
1
2
TABLE
mequiv./l mm
5
l-3
14.3
17.2
60-80%
29.0
43.0
58.3
f 16.1%
68.0
40.0
82.6
r
5.3%
30.0
65.0
82.6
r
5.3%
orally)
GLUCAGON, weight,
were
ALANINE
AND
intramuscularlu).
administered Glucose
Case
mg/dl mg/dl
min
(Duke)
II
GLUCOSE
Case
70
mg/dl
after
10% overnight
concentration
GALACTOSE alanine
TOLERANCE
solution
(0.5
g/kg,
TESTS intravenously)
fasting.
(mg/dl)
0
30
60
Glucagon
69
66
90
Alanine
57
58
57
Galactose
76
70
70
90
120
150
180
95
76
79
50
66
64
56
57
74
73
72
79
Glucagon
68
80
90
81
61
57
57
Alanlne
55
51
55
56
59
61
51
Galactose
41
49
39
42
39
42
39
min
III
GLYCOGEN
CONTENT
AND
ENZYME
ACTIVITIES
Glycogen
G-6-Pase
content
(nmol
(g/loo wet
g
Pi/mg
proteinlmin)
in liver homogenate
Phosphorylase
Amylo-1,6-
(pm01
glucosidase
Pi/
g proteinlmin)
weight)
in liver
(cpmlglmin) in leukocyte
Total
Nonspecific
True
Liver
Leukocyte
Case
1 (U.K.)
7.1
4.57
0.86
3.71
216
50.5
3248
Case
2 (J.M.)
7.9
3.72
2.37
1.35
160
43.9
3940
22.50
1.00
21.0-31.5
2132-9687
Control
(adult)
Control
(N = 10)
21.50
120
and
323
Fig. 1. Lineweaver-Burk case 2.
plots of G+Pase
in liver microsomes
of two patients.
l-
0, case 1; o------O,
Fig. 2. Lineweaver-Burk plots of G-6-Pase in liver microsomes of human control livers. Control 10 days (autopsy); control 2. female adult (biopsy); control 3, male 6 months (autopsy).
TABLE
IV
GLUCOSE-6-PHOSPHATASE The values of the patients
ACTIVITY represent
IN BLOOD
the mean of three determinations.
G-6-Pase activity
Case 1 Case 2 Control (N = 10) Mean ? S.E. Range
PLATELETS
(nmol
glucoselmg
G-6-P
G-6-P (25 mM), fl-GLP (50 mM)
24.1 38.3
18.1 20.6
50.7 ? 5.9 20.1-70.4
25.7 ? 2.4 19.9-39.9
protein/h)
1. female
324
K, for G-6-P of approximately 7.7 mM can be calculated both for the two patients and the controls. The maximum velocities (V,) were 12.5 and 5.3 nmol per mg protein per min for case 1 and case 2, respectively. The control livers yielded values of 91, 133 and 222 nmol per mg protein per min. Nonspecific phosphatase in liver microsomes was not detectable. As shown in Table IV, no significant difference in platelet G-6-Pase activity was found between our patients and. controls when assayed at 25 mM G-6-P with or without 50 mM /3-GLP, though the activities of the patients were close to the low levels of the controls. Discussion Our two patients had clinical and physiological abnormalities similar to those of GSD type I or type III. Usually low fasting blood glucose and mild elevation of blood lactate and cholesterol have been noted in GSD type III. But marked hyperlipemia and nasal bleeding accompanying the abnormalities of platelet function as found in our patients are characteristic manifestations of GSD type I. Also the results of tolerance tests suggest a G-6-Pase deficiency. Reduced G-6-Pase activity, normal activities of phosphorylase and amylo1,6-glucosidase, and elevated glycogen content indicate that both patients are suffering from GSD type I. The slight response to glucagon seems to correlate with low activity of G-6-Pase in liver of both patients [2]. The low activity of G-6-Pase prompted a further characterization of this activity. An apparent K, for G-6-P of 7.7 mM for both of the patients and for controls was found (Fig. 1 and 2) This is of same order as found for human or other mammalian G-6Pase [ 19-201. We could not demonstrate other characteristics of the residual activity because of limiting amounts of material. Further investigations are necessary to show whether this low activity of G-6-Pase represents an incomplete defect or a mutant enzyme. We found a high nonspecific phosphatase activity in the liver of case 2 as found in some cases of GSD type I. The patients with GSD type I reported by Field had high activities of nonspecific phosphatase with @-GLP as a substrate in both liver and intestinal mucosa. These activities were comparable to those as found with G-6-P as a substrate [31-
TABLE
V
PLATELET
GLUCOSE-6.PHOSPHATASE
References
ACTIVITY
G-B-Paw
activity
(nmol
REPORTED gIucose/mg
Controls Linneweh Negishi Schrijver
* The
et al. [91
Substrate
? 432
*
0
-210
*
G-6-P
k 192
*
47
-112
*
G-6-P
0.27
i -i
245
0.05
0.03-
40*
16
20
study
25.1 are calculated
Patients
156
et al. [lo]
values
protein/h)
403
et al. [81
Stormont
Present
et al. [61 et al. [71
Soyama
IN LITERATURE
as 0.6
X 108
?
2.4 platelets
[U-‘4ClG-6-P
*
G-6-P,
0.9.1.7
G-6-P
+ P_GLP
G-6-P
+ ~3-GLP
18.1, are equal
0.07
20.6
to 1 mg of protein.
G-l-P
32.5
Several investigators have reported that G-6-Pase is present in human platelet and that the determination of platelet G-6-Pase activity can be used for the diagnosis of GSD type I [6-lo]. The carrier detection of the disease using platelets was also shown by Soyama et al. [ 71, Negishi et al. [8] and Stormont et al. [ 91. Previous reports are summarized on Table V. The values reported for the control range from 0.27 to 756 nmol per mg protein per hour. Our values for the control are consistent with those of Schrijver et al. [lo], though its activity is much lower than found by Linneweh et al. [ 61, Soyama et al. [7] and Stormont et al. [9]. Their values for the control (245--756 nmol per mg protein per hour) correspond to more than A of G-6-Pase activity (53 nmol Pi liberated per mg protein per min) in normal liver [21]. It seems unlikely that human platelet contained such high G-6-Pase activity, because gluconeogenesis was not present in the way established for liver and kidney. Schrijver et al. demonstrated very low or absent activities of FDPase, PEPCK and PC in human platelets [lo]. In addition the enzyme assay in the cases of Linneweh et al. [6], Soyama et al. [ 71 and Negishi et al. [8] was performed without correction for nonspecific phosphatase, using only G-6-P as a substrate. Stormont et al. [ 91 were using G-1-P for measuring nonspecific phosphatase, and reported this activity was insignificant in more than half of the normal controls while some controls and his patient showed definite levels of nonspecific phosphatase. But Schrijver et al. suggested that the use of G-1-P was not correct because of the presence of glucose phosphomutase in platelet [lo]. Schrijver et al. reported that platelet G-6-Pase activities in his two patients were of the same order of magnitude as those of normal controls when assayed with only G-6-P as a substrate, whereas the enzyme activities assayed in the substrate mixture of G-6-P and fl-GLP were extremely low in the two patients, and concluded that the glucose released from G-6-P in the presence of P-GLP at high concentration could be considered as G-6-Pase activity [lo]. We could not demonstrate any significant difference of platelet G-6-Pase between our patients and control, though the addition of /3-GLP took care of nonspecific phosphatase. In our patients 50 mM fl-GLP might block some of the activity of nonspecific phosphatase but some of this enzyme might remain active upon G-6-P. In view of this, we added increasing concentrations of /I-GLP
TABLE THE
VI EFFECTS
BLOOD The
OF
P-GLP
ON
GLUCOSE-6-PHOSPHATASE
AND
NONSPECIFIC
values
in parentheses
represent
liberated
phosphate
with
only
fl-GLP
as a substrate
tein/h). Substrate G-6-P
25 mM 25 mM
PHOSPHATASE
IN
PLATELETS
Control
Case
1
case
+ @-GLP
+ 12.5
mM
46.7 41.4
21.8 (53.5)
36.4 -
25mM+25mM
41.4
(89.3)
-
-
25mM+50mM
23.8
(143.0)
13.1
17.5
25mM+125mM
21.4
(154.5)
10.9
5.8
25mM+250mM
0.0
(345.0)
0.0
0.0
2
(-01
Pi/mg
pro-
326
to the assay medium. As shown in Table VI, the glucose released from 25 mM G-6-P was decreased by raising the concentration of P-GLP both in two patients and control, and showed about half of that assayed without fl-GLP, when enzyme assay was performed in the substrate mixture of 25 mM G-6-Z’ with 50 mM P-GLP. The liberated phosphate increased by raising concentration of fl-GLP when assayed in only P-GLP as a substrate in control, suggesting the presence of definite levels of nonspecific phosphatase activity. However no glucose production was obtained at a concentration of 250 mM P-GLP. This indicated that the inhibition by high P-GLP of G-6-Pase might occur. Further, we could not deny the possibility that the glucose production (P-GLP 50 mM and 125 mM) indicated the hydrolysis of G-6-P by nonspecific phosphatase, not by G-6-Pase, because it has not yet been confirmed that G-6-Pase is present in human platelet. Our observations indicate that this assay method is not suitable for the proof of diagnosis of GSD type I. We recommend to determine the platelet G-6-Pase activity of many other patients with GSD type I for reaching a definite conclusion. Acknowledgements The authors are grateful to Dr. F.A. Hommes and Dr. J. Schrijver for many discussions and help in the preparation of the manuscript. This investigation was supported by a Research Grant aided by the Ministry of Health and Welfare of Japan for the Research on Handicapped Children, 1977. References 1 Cori, G.T. and Cori, C.F. (1952) J. Biol. Chem. 199.661-667 2 Cornblath, M. and Schwartz, R. (1976) in Disorders of Carbohydrate Metabolism in Infancy (Schaffer. A.J. and Markowitz, M., eds.). pp. 237-260, W.B. Saunders Company, Philadelphia 3 Field, J.B.. Epstein, S. and Egan. T. (1965) J. Clin. Invest. 44.1240-1247 4 SokaI. J.E.. Lowe, C.U., Sarcione, E.J., Mosovich, L.L. and Doray, B.H. (1961) J. CIin. Invest. 40, 364-374 5 Stamm, W.E. and Webb, D.I. (1975) Arch. Intern. Med. 135.1107-1109 6 Linneweh. F., L&r, G.W., Wailer, H.D. and Gross, R. (1962/63) Enzymol. Biol. CIin. 2.188-195 7 Soyama. K.. Schimada, N., Kusunoki. T. and Nakamura, T. (1973) CIin. Chim. Acta 44,327-331 8 Negishi, H.. Morishita, Y.. Kodama, S. and Matsuo. T. (1974) CIin. Chim. Acta 53, 175-181 9 Stonnont. D., Davies, C. and Emmerson. B.T. (1976) Clin. Chim. Acta 71, 303-308 10 Schrijver. J.. Koster. J.F. and Hiilsmann. W.C. (1975) Eur. J. Clin. Invest. 5, 7-14 11 Baginski, E.S.. Foa, P.P. and Zak, B. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U.. ed.), pp. 876-880, Academic Press, New York 12 Fiske, C.H. and Subbarow, Y. (1925) J. Biol. Chem. 66.375-400 13 Hassid. W.Z. and Abraham, S. (1957) in Methods in Enzymology, Vol. III, PP. 34-50. Academic Press, New York 14 Glucomesser direct kit was purchased from Tokyo Zoki Chemical Companv 15 Oka. Y., Matsuda. I., Arashima, S.. Anakura. M.. Mitsuyama. T. and Nambu. H. (1975) NeuropPdiatrie 6.202-209 16 WiBiams, H.E., Kendig, E.M. and Field, J.B. (1963) J. CIin. Invest. 42, 666-660 17 WiIhams. H.E. and Field J.B. (1961) J. CIin. Invest. 40.1841-1845 18 Lowry. O.H., Rosebrough. N.J., Far+. A.L. and RandalI, R.J. (1951) J. Biol. Chem. 193.265-275 19 Nordlie, R.C. (1976) in Gluconeogenesis. its Regulation in Mammahan Species (Hanson, R.W. and Mehhnan. M.A.. eds.), pp. 93-152, Wiley-Interscience. New York 20 Rezani. I. and Duffy, J.L. (1971) Biochem. Med. 5. 237-244 21 Hefferan, P.M. and HowelI. R.R. (1977) Biochim. Biophys. Acta 496. 431-435
